Platelet-free plasma exposed to insolubilized PGE1 (iPGE1) develops the ability to inhibit ADP or epinephrine induced platelet aggregation. This inhibitory activity is independent of changes in platelet cAMP and is mediated through a macromolecular plasma component. We have now identified the responsible plasma component as an altered form of factor Xa. Plasma congenitally deficient in known coagulation factors each inhibited platelet aggregation after exposure to iPGE1 with the exception of factor X deficient plasma. Reconstitution of the deficient plasma with factor X or Xa followed by exposure to iPGE1 restored its ability to inhibit platelet aggregation. Purified bovine factor X or Xa exposed to iPGE1 inhibits platelet aggregation in platelet-rich plasma (PRP). The inhibitory activity of altered factor X can be reversed competitively by factor Xa, but factor X cannot counteract the inhibitory effect of altered factor Xa. Heparin (50 micrograms/ml) added to a PRP neutralizes the inhibitory effect of altered factor Xa (0.02 units/ml) exposed to iPGE1.